Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway

Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-im mortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifie s the mechanism of growth stimulation. TNF-alpha promoted cell cycle progre ssion by increasing expression of HPV-16 E6/E7 RNA5 and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase acti vity was mediated by upregulation of cyclins A and B and decreases in cdk i nhibitors p21(Waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and c yclin A RNAs. To define which components of the EGF receptor signaling path way were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activa ted Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expres sion of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth sti mulation of immortal keratinocytes by TNF-alpha. These studies define a pat hway by which ras mutations, which occur in a subset of cervical cancers, m ay contribute to pathogenesis. Published by Wiley-Liss, Inc.